SUMMARY In this revised, collaborative renewal proposal, we investigate our novel findings regarding the ability of Rho, Ras and Rab GTPases to modulate cytoskeletal and membrane apical-basal polarization as well as vesicle trafficking to the apical membrane surface to control human endothelial cell (EC) tubulogenesis. Using state-of- the art in vitro and in vivo approaches, we have demonstrated a fundamental role for Cdc42 during this process with human ECs and during mouse vascular development and blood vessel growth. Inactivation of Cdc42 function leads to disruption of EC polarization in vivo and in vitro that results in a failure to properly form or organize EC lumen and tube networks. In addition, RhoA inactivation in vivo vs. in vitro does the opposite of Cdc42, where increased lumen formation occurs. Importantly, we have shown that Rasip1 and its binding partner, Arhgap29, function together to suppress RhoA signaling to allow EC tube formation to occur. To further investigate this process, we have developed a highly defined approach to elucidate when and where particular molecules and signaling pathways act. We can directly assess whether individual molecules or signals separately control intracellular vacuole formation, cytoskeletal polarization, vacuole trafficking along the tubulin cytoskeleton toward the apical surface, or vacuole fusion in the subapical region to create the apical membrane. We can perform these studies due to our ability to regulate the expression or activity of key molecules coupled with the ability to visualize intracellular vacuoles, the polarized cytoskeleton and the apical surface (in static or real-time video images) using EC apical labels such as GFP-caveolin1. Together, these results provide a molecular road map to elucidate how ECs change shape, polarize, reorient junctions, and move membranes to the apical surface; all with the ultimate goal of forming functional tubes that carry blood, a capacity essential for blood vessel formation, tissue viability, and tissue development. Here, we test the hypothesis that EC lumen formation depends on GTPase signaling cascades that promote the intracellular transport of membranes to the apical surface and polarized subcellular recruitment of critical effectors. We propose three specific aims to further investigate these novel insights into the fundamental process of EC tubulogenesis in vivo and in vitro and they are: Aim #1. To elucidate the underlying molecules and mechanisms responsible for suppression of RhoA at the apical membrane in ECs; Aim #2. To investigate Rab GTPase control of vacuole/vesicle formation, trafficking and fusion, leading to polarized EC apical membrane assembly and lumen formation; Aim #3. To investigate the specific roles of key upstream guanine exchange factors (GEFs) vs. GTPase activating proteins (GAPs) regulating Cdc42, Rac, k-Ras and Rap1b during EC lumen formation.